Experimental design (Fig. 1)

Experimental protocol. Isch ischemia only, IPreC ischemic preconditioning, IPostC ischemic postconditioning, RIC remote ischemic conditioning, LAD left anterior descendent coronary artery, TTC triphenyl tetrazolium chloride, MRI magnetic resonance imaging

Domestic female pigs (25–35 kg; genotype: DanBred hybrid, purchased from the University of Kaposvár, Hungary) were kept according to the Big Dutchman principles. The animals were fed with a pregnant sow diet containing a low energy and balanced protein level produced by Dalmand co. Ltd. They further underwent regular veterinarian check-ups, and only healthy animals were selected for the study. Furthermore, before percutaneous intervention, transthoracic echocardiography, and angiography was served as a baseline screening method to exclude animals with abnormal coronary anatomy or myocardial disease.

The pigs were block randomized [22] into four groups: ischemia only (Isch; n = 17), IPreC (n = 12), IPostC (n = 14) and RIC (n = 17). Three animals died during myocardial ischemia (Isch: 1; IPreC: 1; IPostC: 0; RIC: 1) and 4 during reperfusion due to therapy resistant malignant ventricular rhythm disturbances (Isch: 2; IPreC: 1; IPostC: 1; RIC: 0). Additionally, 3 animals were excluded due to procedural technical reasons. The final case numbers were 14, 9, 12 and 14 in Isch, IPreC, IPostC and RIC groups, respectively. According to the current ESC STEMI guidelines [23, 24], the pigs were pretreated with loading doses of 250 mg acetyl salicylic acid and 300 mg clopidogrel with maintenance doses of 100 and 75 mg pro day, respectively. Although 600 mg dose of clopidogrel has been shown to superior over a 300 mg dose in myocardial necrosis reducing effects [25], our experimental protocol has been planned to minimize the effect of possible confounding factors such as clopidogrel [26]. Animals were sedated with 12 mg/kg ketamine hydrochloride, 1 mg/kg xylazine, and 0.04 mg/kg atropine intramuscularly after an overnight fast. Anesthesia was induced by inhalation of isoflurane (2–2.5 vol%). Animals were intubated endotracheally and anesthesia was maintained by inhalation of an isoflurane oxygen mix (2–2.5 vol% and 3 L/min). Magnesium sulphate (4.06 mEq diluted in 10 mL, in every 60 min) and a continuous amiodarone infusion (300 mg diluted in 500 mL saline) were being administered throughout the procedure via an ear vein. Sheaths were inserted into femoral artery and femoral vein to further have entry routes for the catheterization, and 5000–5000 IU heparin was administered via each sheath. Cardiac function was assessed with echocardiography. Baseline hemodynamics were recorded, and selective angiography of the left coronary artery was performed. After the analysis of the baseline angiogram, a balloon catheter (2.75 mm diameter, 8 mm length) (Abbott Vascular) was placed in the mid part of the left anterior descending coronary artery (LAD) after the origin of the 2nd diagonal branch. For induction of AMI, the intracoronary balloon was inflated with 5 atm for 90 min, followed by deflation of the balloon, resulting in reperfusion (3 h or 3 days) which was confirmed by coronarography. In IPreC group, LAD was occluded by the inflation of the balloon at 5 atm 3 times for 5 min followed by 5 min of reperfusion, while in other animals the balloon was left deflated for 30 min [27]. Then LAD was occluded by inflating coronary balloon, which was confirmed by coronarography. RIC was performed by 4 cycles of 5 min occlusion and 5 min reperfusion of the femoral vessels by tightening and releasing of a snare around the right hind limb starting at the 50th min of LAD occlusion [9]. We verified the hind limb ischemia in three ways: (1) Apparent lividity during ischemia and pronounced hyperemia during reperfusion was observed distal to the occlusion. (2) In each animal, a superficial femoral artery was cannulated distal to the occlusion, and blood pressure was measured during the intervention. The minimum of 30/30 mmHg blood pressure was achieved while the wire was tightened around the hind limb. (3) In one particular animal, we performed a femoral angiography before and during hind limb ischemia as well [Additional file 1: Video_S1.avi (before), Additional file 2: Video_S2.avi (during hind limb ischemia)]. IPostC was initiated within 1 min after the termination of 90-min index ischemia and was performed by applying 6 cycles of 30/30 s LAD occlusion and reperfusion [28]. The interventional cardiologist was not blinded during the investigation due to the nature of the procedure: IPreC and IPostC were achieved by inflating a balloon in the LAD, which required an unblinded interventional cardiologist. Nonetheless, the interventional cardiologist was not aware of the allocation until the initiation of the experimental intervention (e.g. right after balloon deflation if the animal was in IPostC group). After reperfusion was initiated 5000 IU heparin was given intracoronary. Final reperfusion was confirmed with coronarography and the catheters were removed. Ten min after the initiation of reperfusion, the hemodynamic data was recorded again. Anesthesia was either maintained for 3 h or in case of 3 days reperfusion, wounds were closed and anesthesia was terminated by the withdrawal of isoflurane. Analgesia was applied by intramuscular injections of 1 g metamizole. An antibiotic cocktail (100 mg benzathine benzylpenicillin, 100 mg procaine benzylpenicillin, 200 mg dihydrostreptomycin-sulphate) was given i.m. before recovery.